Two novel advances of this and associated laboratories underlie the application of this research to DNA vaccines against Ebola and dengue viruses, and potentially other emerging viruses: (1) The coupling of the targeting sequence of lysosomal membrane proteins to the protein antigen encoded by DNA vaccines; we have shown this to enhance markedly the immune response to several antigens, presumably by directing the antigen to the major histocompatability class II (MHC II) pathway for processing and presentation of antigenic epitopes to the helper T cell arm of the immune response mechanism. (2) A non-viral system for the in vivo delivery of genes as a DNA-gelatin nanosphere coacervate as a means to deliver the DNA vaccine to antigen presenting cells and for the co-delivery of targeting ligands and cytokines that augment the immune system response. The principle goals of the proposed research are to perfect these systems with DNA vaccines encoding Ebola and dengue virus proteins and to prove the feasibility of these approaches for possible application to other emerging viruses. This research will be conducted in collaboration with other laboratories that maintain animal model systems for Ebola and dengue virus challenge. The research design is, first, to prepare DNA plasmid constructs encoding chimeric Ebola and dengue virus protein antigens containing the signal and targeting sequences of the LAMP lysosomal membrane protein. The immune response of mice vaccinated with these plasmids will be compared to that of mice vaccinated with DNA encoding the wild type protein antigens. These experiments will also compare the response of mice to vaccination with naked DNA as compared to nanosphere DNA. The experiments will include studies of DNA dose, route of administration, kinetics of vaccination, and duration of the immune response. Additional experiments will analyze the effect of co-delivery of cytokines, including interleukins 2 and 4, granulocyte-macrophage colony stimulating factor, and gamma interferon. Additionally, the immune response to of antigens targeted to the lysosomal membrane will be compared to the effect of DNA encoding the protein antigens coupled to the CTLA4 ligand that binds to the B7 protein of antigen presenting cells, and to the response to nanosphere DNA directed to antigen presenting cells.